1. Field of the Invention
The present invention relates to a cell culture apparatus useful for the cultivation of animal, insect, micobial, or plant cells in industrial or medical applications.
2. Background--Description of Prior Art
In vitro cell culture is an important operation for the production of pharmaceuticals such as antibodies, cytokines, viral gene vectors, and for cell therapy. Many devices have been developed for cell culture. These can be divided into two major categories: 1) small-scale devices with a culture volume of 1 to 2 liters; and 2) bioreactors with operating volumes of 1 to 10,000 liters. The small-scale devices are limited to a few liters in volume because these devices rely on surface oxygen transfer to provide the required aeration for cells to grow and make the desired product. Such devices include spinner flasks, T-flasks, roller bottles, and gas-permeable bags. Their limitation on culture volume is very serious because many applications require 10 to 100 liters of cell culture in order to produce sufficient quantity of the product of interest. Using conventional small-scale equipment, this requires the tedious production of multiple batches.
Traditionally, production of larger volumes has required the use of bioreactors. Bioreactors, however, require elaborate mechanical systems to provide aeration and mixing. Control systems are required to sterilize the equipment and regulate temperature, pH and dissolved oxygen levels. This makes a bioreactor expensive to acquire, install, maintain and operate. Extensive training is required to operate these bioreactors without contamination. For these reasons bioreactors are only used in large industrial and academic environments. The conventional cell culture bioreactor is a stirred tank that has been adapted from microbial cultivation by the addition of low-shear mixers and more gentle aeration systems. (Armstrong et al U.S. Pat. No. 4,906,577) and Morrison U.S. Pat. No. 5,002,890.
These improvements are relatively minor and very little real innovation has been made since the 1960's.
In examining the many cell cultivation device patents it is apparent that most have been commercial failures due to: 1) failure to provide an appropriate environment for cell cultivation, and 2) excessive complexity. The patent by Lu et al (U.S. Pat. No. 5,443,985) is an example of the first problem. Here, the inventors after discussing the detrimental effects of bubbles in cell culture propose a cultivation device in which both agitation and aeration is accomplished solely by bubbling air into the device. An example of excessive complexity with little benefit is U.S. Pat. No. 5,057,429 (Watanabe et al). Here a gas permeable bag is used to diffuse oxygen and nutrients to the cells. A second inner bag contains the cells. An elaborate agitation apparatus with complex motion is described. However, the device is essentially oxygen transfer limited by the surface to volume ratio of the gas permeable surface. It is thus not possible to scale the unit beyond a few liters. A more or less similar result is achieved using the static gas-permeable bag described by Matsumiya et al (U.S. Pat. No. 5,225,346). Of course, as with all devices that rely on gas-permeable surfaces, scale-up is severely restricted. Amoit et al (U.S. Pat. No. 5,202,254) describe improvements to the hollow fiber bioreactor. However, their ideas do not address the fundamental problems of the hollow fiber devices. These are formation of oxygen and nutrient gradients in the system; lack of mixing; difficulty in sampling and the complexity of the support system (oxygenator, pump, instrumentation and circulation tubing). A recent patent by Dziewulski et al (U.S. Pat. No. 5,135,853) continues the trend in the field to ever more complex cultivation devices by introducing a three-compartment bioreactor.
Instead, it is necessary to reduce the complexity of the cultivation apparatus by understanding and exploiting the unique characteristics of cell cultivation. For design to be successful it must therefore:
Eliminate gas bubbles which are now known to cause cell damage. PA1 Eliminate high local shear caused by rotating mixers. PA1 Provide sufficient mixing to provide a homogeneous environment, prevent cell settling, and promote as transfer. PA1 Provide a sterile, disposable cultivation vessel to reduce labor cost and the need for steam sterilization. PA1 Not use permeable membranes or static surface aeration in order to facilitate scale-up. PA1 Reduce mechanical and instrumentation complexity to a minimum.
The present invention will provide a new and improved method for culturing cells in vitro that achieves all these criteria, and overcomes all the aforementioned prior art limitations.
Objects and Advantages
Key objects and advantages of the present invention are:
(a) Reduces the cost of a cell culture bioreactor by a factor of 100 compared to conventional glass and stainless steel stirred tank bioreactors. PA0 (b) Provides a non-invasive means of agitation that reduces mechanical complexity and possibility of contamination. This mode of agitation minimizes local high shear fields that cause cell damage. PA0 (c) Improves cell growth and productivity by providing a bubble-free means of aeration that minimizes damage to cells caused by bubbles and foam formation. PA0 (d) Provides an easy to operate culture device suitable for industrial and hospital environments. It eliminates the need for labor-intensive cleaning, preparation and sterilization, typical of conventional stainless steel bioreactor equipment by providing a pre-sterilized disposable one-use device. The low mechanical complexity of the present invention reduces operating and maintenance costs. PA0 (e) Provides complete isolation of cells allowing cultivation in a non-aseptic environment, and is also useful for the culture of pathogens, viruses and other organisms requiring a high degree of containment. PA0 (f) Can be operated with widely varying culture volume. This allows for seed buildup within the culture vessel by adding media without the need for seed bioreactors and contamination-prone vessel-to-vessel transfers.
Further objects and advantages of my invention of my invention will become apparent from a consideration of the drawings and ensuing description.